Pre-mRNA splicing is an essential step in gene expression. It involves precise excision of introns and joining of exons from primary transcripts in the nucleus to generate mature mRNA, the template for protein synthesis in the cytoplasm. Many genes express multiple mRNA and protein isoforms by alternative splicing of their primary transcripts. The catalyst for splicing is the spliceosome, a macromolecular machine composed of five small RNAs and numerous proteins, which undergo dynamic assembly with each exon-intron-exon substrate. Prior to translation, the mature mRNA is subject to a quality-control pathway, known as nonsense- mediated decay (NMD). NMD degrades mature mRNAs with a premature termination codon (PTC). In mammals, the difference between premature and normal PTCs is established in relation to their position and distance from the last exon-exon junction. This reflects splicing-dependent loading of an exon-junction protein complex (EJC). PTCs arise by nonsense, frameshift, or splicing mutations, as well as being present in some normal alternatively spliced mRNA isoforms. The overall goal of the proposed research is to study selected aspects of mammalian pre-mRNA splicing, with an emphasis on the recognition of the splice sites by selected human splicing factors. Specific aspects of splicing-dependent mRNA surveillance will also be investigated. The specific aims are: 1. To study the mechanism of 5'-splice-site selection;2. To study RNA-binding proteins involved in 3'-splice-site recognition;3. To study the links between pre-mRNA splicing and NMD;4. To study exon-recognition defects in disease-susceptibility genes. These objectives will be achieved through a combination of biochemical approaches using cell-free systems, purified components, and recombinant proteins together with molecular and reverse-genetic approaches in cell culture, bioinformatics analysis of the genome, and methods to modulate gene expression. Aberrant splicing due to mutations in intron-containing genes is a frequent cause of many human genetic diseases. Information gained from these basic studies already informs genetic counseling and phenotypic-risk assessment in the clinic, yet there is much room for improvement in predicting genotype-phenotype correlations that involve aberrant splicing or NMD. Mutations in splicing factors are also associated with inherited diseases, such as retinitis pigmentosa, and with cancer. For example, the gene encoding the splicing factor SF2/ASF is a proto-oncogene that can be activated by gene amplification in breast cancer. Drugs are being developed to correct aberrant splicing, e.g., through antisense methods, small molecules, or bifunctional modulators. Some of these approaches are already being tested in clinical trials for muscular dystrophy and spinal muscular atrophy, while others are still in the pre-clinical stages. The proposed studies, which are aimed at understanding basic cellular mechanisms of gene expression, may provide the basis for future development of new or improved diagnostic tools and therapeutic agents. PROJECT NARRATIVE - Pre-mRNA splicing and mRNA quality-control are key steps in gene expression. Mutations that affect these processes in specific genes, or that alter the properties or levels of the factors involved in these pathways, underlie many genetic diseases and cancer predispositions. The proposed studies are aimed at understanding basic cellular mechanisms of gene expression, and may provide the basis for developing or improving diagnostic tools and therapeutic agents for these diseases.